1. Field of the Invention
Present invention relates to a method of semi-solid state fermentation for production of surfactin from a high-yield mutant strain of Bacillus subtilis subsp.
2. Description of the Prior Art
Biosurfactants are secondary metabolites that are produced by microorganisms under certain conditions and contain both hydrophilic and hydrophobic structures. In addition to having the same or similar physical and chemical properties as the chemically synthesized surfactants, biosurfactants also have a number of other characters such as complex structure, high specificity, low toxicity, biodegradable, environmental friendly and can be produced by fermentation of cheap agricultural by-products. Moreover, certain biosurfactants also have antibacterial, anti-viral and anti-tumor pharmacological effects. Most lipopeptide biosurfactants (LPBSs) have anti-microbial effect and are also called antibiotics. At present, the major LPBSs identified include surfactin, fengycin, iturin, bacillomycin, mycosubtilin and plipstatin, etc. By far, biosurfactants have been widely used in the fields of cosmetics, food, oil and pharmaceutical industry.
In 1968, Arima et. al discovered a lipopeptide biosurfactant produced by Bacillus subtilis ssp. with high activity and named it surfactin. The yield of surfactin produced by Arimaet. al was only 0.04-0.05 g/L. In 1981, Cooper et. al used a basal mineral salt medium to grow Bacillus subtilis ATCC21332 and collected the foam and isolated surfactin, the yield was 0.78 g/L, Mulligan et. al in 1989 found a UV-induced mutant strain of Bacillus subtilis ATCC51338 and its surfactin yield was 1.124 g/L. In 1997, Kim et. al cultured Bacillus subtilis C9 under a condition with limited oxygen in an improved Cooper medium and its surfactin yield was 7.0 g/L. By 2000, Wei Yu-Hong et. al increased the yield of surfactin to 3.5 g/L by using a mineral enhanced salt medium and controlling the pH.
The main method for producing surfactin from Bacillus subtilis at present is liquid fermentation because the fermentation parameters that can be controlled are relatively more for liquid fermentation; nonetheless, the major controlling parameters are the ingredients of the media, exploration of fermentation conditions and screening of the high-yield strain. Studies have indicated that different carbon sources in the media indeed will affect the yield of surfactin (Lang, et al., 1999; Wei, et al., 2004; Wei, et al., 2005). Glucose can be used as the carbon source and be utilized by Bacillus subtilis; yet, excess glucose will result in reduced pH which then leads to reduced yield of surfactin. Therefore, production of surfactin is closely related to the pH of the media (Yeh, et al., 2005). Addition of long-chain carbon also helps increase the yield of surfactin (Ghribi and Ellouze-Chaabouni, 2011). Different nitrogen sources have two effects: 1. yield: adjusting the formula of the medium proposed by Cooper in 1981 by changing its nitrogen source and growing the bacteria under the anaerobic condition increases the yield of surfactin to 7 g/L (Kim, et al., 1997); 2. the structure of surfactin: addition of different hydrophobic amino acids to the medium changes the structure of surfactin (Peypoux, et al., 1992). When the concentrations of divalent and trivalent iron in the medium are increased from μg to mg, the total yield of surfactin produced by Bacillus subtilis ATCC 21332 will increase. On the other hand, addition of iron ions will lead to reduced pH of the medium and Bacillus subtilis will stop producing surfactin when the pH is lower than 5. Hence, addition of iron ions must be coordinated with adjustment of pH in order to facilitate surfactin production (Wei and Chu, 1998; Wei and Chu, 1998).
Moreover, addition of manganese ions to the media can significantly increase the yield of surfactin while not affecting the growth of Bacillus subtilis (Wei and Chu, 2002). Furthermore, growing Bacillus subtilis in a medium without metal ions of magnesium, potassium, manganese and iron was found to significantly decrease the yield of surfactin as well as the rate of growth, indicating the four ions are essential for the culturing process in spite of the fact that they are trace elements (Wei, et al., 2007). Some researchers pointed out, in addition to the ingredients of media, addition of solid carriers to media can also effectively increase the yield of surfactin. Because activated carbon is not easily degraded during the process of fermentation and can be easily dissociated from a medium, using activated carbon as the carrier to increase the number of cells so as to delay the cells from entering the stagnation stage can improve the yield of surfactin (Yeh, et al., 2005).
The disadvantage of liquid fermentation is that addition of a defoaming agent during the process of fermentation is required, which consequently leads to difficulties in purification. Thus, the method of fermentation needs to be changed in order to increase the yield of surfactin.
Except for liquid fermentation, other available methods include solid-state fermentation which is one important fermentation method in the fermentation industry due to low costs of its substrates. Previous literatures indicated the groups with low solid contents after fermentation of Bacillus subtilis subsp ATCC 21332 by using the waste generated from potato processing have higher yields of surfactin when compared with the groups with high solid contents (Nitschke, et al., 2004). Fermentation of the by-product and wastes of tofu processing by using Bacillus subtilis subsp MI113 at 37° C. for 48 hrs produces 2 g/kg of surfactin (Ohno, et al., 1995) and the surfactin produced from soybean fermentation using Bacillus polyfermenticus KJS-2 shows anti-microbial activity and its minimum inhibitory concentration (MIC) is 0.05 mg/mL (Kim, et al., 2009).
The disadvantages of solid-state fermentation include: 1. low water content limits the transmission rate of substances and energy and the heat generated during fermentation cannot be easily removed and thus resulting in a gradient of nutrition which then causes unbalanced fermentation; 2. fermentation parameters are not easily detected (e.g. pH value and biomass), poor reproducibility; 3. substrate stirring is not easy which may easily cause destroyed environment for microbial growth; 4. the time of solid-state fermentation is longer than liquid fermentation. Surfactin is one of the most active biosurfactants and has attracted much attention since its discovery. However, due to its low yield and high product costs, industrial application of lipopeptide biosurfactants have been unsuccessful. By far, large-scale industrial production of surfactin is still impossible. Hence, many inventors have devoted themselves to increasing the yield of surfactin. Moreover, transformation from laboratory techniques to industrial production is a very difficult issue due to instability of the bio-fermentation process.
How to reduce the production costs is the main goal of this study, which requires selection and culturing the high-yield strain as well as suitable and cheap culture media in order to reduce the fermentation costs and increase the unit yield. In addition, recycling of the fermented substrates and developing high value-added products are just the technical issues this study intends to address.
The purpose of the invention is to overcome the deficiencies of prior art and to provide a commercial preparation method for producing lipopeptide biosurfactants. This method adopts semi-solid state fermentation to improve part of the disadvantages of solid-state fermentation. Because the water content in semi-solid state fermentation is higher than that of solid-state fermentation, the problems of transmission rate of substances and energy, heat generated during fermentation and longer fermentation time can all be solved. Moreover, the yield of surfactin by semi-solid state fermentation can be increased by changing the basic conditions for fermentation such as water content, substrates for fermentation, and addition of oils as well as amino acids to supplement the carbon and nitrogen sources. This production technology offers high yield, high efficiency, short fermentation cycle and easy manufacturing process which can significantly reduce the production costs and is suitable for industrial application as well as large-scale production; moreover, it is very helpful for promotion and application of lipopeptide biosurfactants.